Over the past several years, quantitative in vitro studies of kinetics and inhibitors have firmly established the general characteristics of sugar and amino acid transport in the intestine, e.g., energy dependence, competition, saturation, ionic dependence, and sensitivity to specific inhibitors. However, if we are to fully understand this transport process, the basic cellular and molecular mechanisms underlying this characteristic must be explained. This proposal focuses on the in situ molecular properties of the uphill entry process. Specific efforts will be directed toward determining first the kinds of chemical groups of brush border-membrane which are specifically required for sugar and amino acid absorption, their membrane density and reactivity and secondly to isolate the membrane proteins containing these chemical groups. The experimental approach will be to determine how selected chemical modifications of sugars, amino acids and membrane structure affect epithelial cell entry. The nature of these effects will be ascertained from analysis of inhibition kinetics and perturbation of influx kinetics using theoretical models of irreversible inhibition of transport sites exhibiting Michaelean kinetics. Sugar and amino influxes will be measured directly by mucosal exposures to radiolabelled substrates. Tissue uptake of inhibitors will be determined either by radio tracer analysis, autoradiographically or colorimetrically. The membrane proteins which have reacted with inhibitor will be isolated with inhibitor using antibody columns specific to the inhibitor hapten.